The Effect of Cilostamide on Gap Junction Communication Dynamics, Chromatin Remodeling, and Competence Acquisition in Pig Oocytes Following Parthenogenetic Activation and Nuclear Transfer1

Abstract

In the pig, the efficiency of in vitro embryo production and somatic cell nuclear transfer (SCNT) procedures remains limited. It has been suggested that prematuration treatments (pre-IVM) based on the prolongation of a patent, bidirectional crosstalk between the oocyte and the cumulus cells through gap junction mediate communication (GJC), with the maintenance of a proper level of cAMP, could improve the developmental capability of oocytes. The aim of this study was to assess: 1) dose-dependent effects of cilostamide on nuclear maturation kinetics, 2) the relationship between treatments on GJC functionality and large-scale chromatin configuration changes, and 3) the impact of treatments on developmental competence acquisition after parthenogenetic activation (PA) and SCNT. Accordingly, cumulus-oocyte complexes were collected from 3- to 6-mm antral follicles and cultured for 24 h in defined culture medium with or without 1 μM cilostamide. GJC functionality was assessed by Lucifer yellow microinjection, while chromatin configuration was evaluated by fluorescence microscopy after nuclear staining. Cilostamide administration sustained functional coupling for up to 24 h of culture and delayed meiotic resumption, as only 25.6% of cilostamide-treated oocytes reached the pro-metaphase I stage compared to the control (69.7%; P < 0.05). Moreover, progressive chromatin condensation was delayed before meiotic resumption based upon G2/M biomarker phosphoprotein epitope acquisition using immunolocalization. Importantly, cilostamide treatment under these conditions improved oocyte developmental competence, as reflected in higher blastocyst quality after both parthenogenetic activation and SCNT.

Transcriptomic signature of the follicular somatic compartment surrounding an oocyte with high developmental competence

“…Using cAMP-elevating agents such as forskolin and IBMX for in vitro pre-maturation, GJC is enhanced and helps improve oocyte competence 40 . The enhanced GJC affects oocyte chromatin remodelling and transcription 41 , oocyte metabolism 8 , and accumulation of intra-oocyte GSH 42 , which are necessary for the acquisition of developmental competence. Thus, enhancement of GJC by elevating and/or sustaining cAMP concentration in cumulus cells subjected to FSH-priming may also be a reason for improved oocyte competence.…”

Natriuretic peptides improve the developmental competence of in vitro cultured porcine oocytes

“…The culture medium was divided into two portions for different assays. Briefly, the PMC medium was composed of basal medium plus BNP (PMC- BNP) or CNP (PMC- CNP) at a final concentration of 100 ng/mL or cilostamide (10 μM) [24] in the culture medium. All cultures were carried out in a humidified atmosphere at 38.5 °C with 5% CO 2 in air.…”

Fatores anatomofisiologicos que afetam a qualidade oocitária em bovinos

“…Além disso, o efeito da suplementação com progesterona durante o desenvolvimento do folículo pré--ovulatório potencializa o estabelecimento do corpo lúteo, aumentando consequentemente a concentração de progesterona circulante durante o desenvolvimento embrionário inicial e incrementando de forma particular a elongação do embrião em fase de pré-implatação (Lonergan & Forde 2014, Maillo et al 2014, Wiltbank et al 2014 Ainda, tem sido reportado que o intercâmbio entre o oócito e as células do cumulus é fator chave para o sucesso na maturação, fertilização e início de desenvolvimento embrionário, o que coloca o estudo das células do cumulus como prioritário para predizer a competência oocitária (Tanghe et al 2002). Adicionalmente a manutenção de um acoplamento funcional adequado entre oócito e cúmulus parece ser crucial para sustentar uma ordenada condensação da cromatina in vitro (Dieci et al 2013, Luciano et al 2014. Substâncias regulatórias produzidas pelo oócito têm importantes funções no metabolismo das células do cúmulos e os produtos dessas células somá-ticas participam ativamente do crescimento e maturação de oócitos (Landim-Alvarenga & Maziero 2014).…”